PROJECT SUMMARY There is an urgent need for novel therapeutics to treat drug addiction. One potential novel drug target that plays a key role in this devastating disorder is ?FosB. ?FosB accumulates in highly specific regions of the brain (in particular the nucleus accumbens) in response to cocaine or other drugs of abuse. ?FosB mediates increases in drug-seeking behavior seen after prior drug exposure. As a transcription factor, ?FosB regulates the expression of many genes crucial to drug addiction, including the AMPA glutamate receptor subunit GluA2 and cyclin-dependent kinase 5 (Cdk5). ?FosB can both repress and activate gene transcription, but the molecular basis of this dual action is not known. One explanation is that ?FosB forms both heterodimers with JunD as well as homodimers with itself, and that these two ?FosB-containing species differentially regulate gene transcription. We seek to validate the therapeutic potential of ?FosB, and to delineate its molecular mechanisms. To this end, our goal is to leverage compounds that target ?FosB species in vivo. We hypothesize that, by regulating ?FosB with small molecules, we can exploit ?FosB to strategically regulate key genes and overcome harmful neuronal and behavioral adaptations induced by chronic cocaine. Our approach is to develop potent in vivo chemical probes that target ?FosB and discriminate between ?FosB homodimers and heterodimers. We demonstrated with first generation scaffolds that pharmacologically targeting ?FosB elicited biological and behavioral responses in mice chronically treated with cocaine. We have now identified new scaffolds with more drug-like properties, but low micromolar activity, which we have validated in vitro. We propose to: 1) improve the potency of our probes through chemical optimization and iterative testing, 2) demonstrate that our compounds directly bind ?FosB and reveal their mechanism-of-action using structural biology, and 3) measure the impact of our probes in vivo both on the behavioral responses to cocaine, and on the transcription of GluA2 and Cdk5. The rationale for this proposal is that improved probes will enable us to test the therapeutic potential of ?FosB as a viable drug target to ameliorate aspects of drug addiction. In addition, our improved probes will enable us to delineate aspects of ?FosB in vivo (in particular the role of ?FosB homodimers vs. heterodimers). This proposal is innovative because it will yield chemical tools for a novel and non-traditional putative therapeutic target for which no probes are currently available. Importantly, we will be able to test whether ?FosB can be used as a conduit to safely regulate specific genes that maintain the addicted state by harnessing the highly region-specific accumulation of ?FosB in the nucleus accumbens in response to drugs of abuse. Furthermore, our probes will enable us to reveal completely new mechanistic information on ?FosB which cannot be easily gained using current techniques. This information is very significant because it could reveal completely novel strategies to treat drug addiction.